Maria Angela Bagni

Absence of mechanical evidence for attached weakly binding cross‐bridges in frog relaxed muscle fibres.

1. Passive force responses to ramp stretches at various velocities were measured in intact and skinned single muscle fibres isolated from the lumbricalis muscle of the frog. Force was measured using a fast capacitance transducer and sarcomere length was measured using a laser light diffraction technique at a point very close to the fixed end so as to avoid effects of fibre inertia. Experiments were performed at 15 degrees C with sarcomere length between 2.13 and 3.27 microns under high (170 mM) and low (20 mM) ionic strength. 2. The analysis shows that the force response is the sum of at least three components: (i) elastic (force proportional to the amount of stretch), (ii) viscous (force proportional to rate of stretch), and (iii) viscoelastic (resembling the response of a pure viscous element in series with an elastic element). 3. The amplitude of all these components increased progressively with sarcomere length in the whole range measured. 4. A further component, attributable to the short‐range elasticity (SREC), was present in the force response of the intact fibres. 5. The amplitude of the force response decreased substantially upon skinning at high ionic strength but increased again at low ionic strength. The SREC was completely abolished by skinning. 6. None of the components of the force response was found to have the properties expected from the previously postulated ‘weakly binding bridges’.

Development of stiffness precedes cross-bridge attachment during the early tension rise in single frog muscle fibres.

1. Force responses to ramp stretches were recorded in single muscle fibres isolated from the lumbricalis muscle of the frog. Stretches were applied at rest and at progressively increasing times after a single stimulus. 2. The increase of fibre stiffness that precedes tension development has a ‘static’ component that accounts for the whole fibre stiffness increase during the latent period and at very low tension at the beginning of the twitch. 3. Static stiffness increase was not affected by 2,3‐butanedione‐2‐monoxime, a drug that almost completely inhibited twitch tension. 4. Static stiffness increased approximately 5‐fold as the sarcomere length was increased from 2.1 to 2.84 microns. 5. These results suggest that static fibre stiffness increase is not attributable to the formation of non‐force‐generating cross‐bridges.

Tension and stiffness of frog muscle fibres at full filament overlap.

SummaryStiffness measurements in activated skeletal muscle fibres are often used as one means of estimating the number of attached crossbridges on the assumption that myofilament compliances do not contribute significantly to the fibre compliance. This assumption was tested by studying the effects of sarcomere length on fibre stiffness in the plateau region of the length-tension diagram (from 1.96 to 2.16μm sarcomere length in the tibialis anterior muscle of the frog). Lengthening of the sarcomere across this region in fact, produces only an increase in the proportion of actin filament free from cross-bridges without altering the amount of effective overlap; no change in fibre stiffness is therefore expected if actin filaments are perfectly rigid. The results show that while tetanic tension remained constant within 1.5%, as the sarcomere length was increased from 1.96 to 2.16μm fibre stiffness decreased by about 4%, indicating that a significant proportion of sarcomere compliance is localized in the actin filaments. A simple model based on the sliding filament theory was used in order to calculate the relative contribution of actin filaments to fibre compliance. In the model it was assumed that fibre compliance resulted from the combination of crossbridge compliance (distributed over the overlap zone) in series with thin filament and tendon compliances. The calculations show that the experimental data could be adequately predicted only assuming that about 19% of sarcomere compliance is due to actin filament compliance.

Effects of 2,3-butanedione monoxime on the crossbridge kinetics in frog single muscle fibres.

SummaryThe effects of 2,3-butanedione monoxime (BDM) on contraction characteristics were studied at 5‡C in single intact fibres isolated from the tibialis anterior muscle of the frog. The force-velocity relation was determined using the controlled-velocity method in either whole fibres or short fibre segments in which sarcomere shortening was measured by a laser light diffraction method. It is shown that 3mm BDM decreases the speed of rise and the amount of tetanus tension, reduces the maximum velocity of shortening and increases the curvature of the force-velocity relation, as well as the value for the stiffness to tension ratio. BDM also slowed down the redevelopment of tetanus tension after a period of unloaded shortening both in fixed-end and in length-clamp conditions. In normal and in BDM-treated fibres length-clamping increased the speed of the initial rise of tetanus tension but not that of the recovery after shortening. The observed force-velocity data points were fitted by the Huxley (1957) equation. It was found that BDM produces a conspicuous decrease of the rate constant for crossbridge attachment. This effect, and also a reduction of the force per crossbridge, are responsible for the depression of the contractile characteristics produced by BDM.

Plateau and descending limb of the sarcomere length-tension relation in short length-clamped segments of frog muscle fibres

1. The relation between sarcomere length and tetanic tension was determined at 10‐12 degrees C for 70‐80 microns long segments of single fibres isolated from the tibialis anterior and semitendinosus muscles of the frog. Measurements of segment striation spacings were performed during fixed‐end or length‐clamp contractions by means of a laser light diffractometer. 2. At sarcomere lengths of around 2.10 microns tetanic tension rose promptly to a steady plateau, independent of the recording conditions. At greater sarcomere lengths under fixed‐end conditions the tension rise occurred in two distinct stages: an initial rapid rise followed by a much slower creep. The tension creep was entirely abolished in length‐clamp contractions. 3. The sarcomere length‐tension diagram of length‐clamped segments of tibialis anterior fibres exhibited a definite flat region between about 1.96 and 2.16 microns where tension varied by less than 1.5%. The highly linear descending limb reached zero tension at about 3.53 microns. The shift to the left by about 0.10 microns, with respect to the length‐tension diagram of length‐clamped segments of semitendinosus fibres, may be tentatively explained by assuming that thin filament lengths vary in different muscles. 4. The results are in agreement with those of a previous work by Gordon, Huxley & Julian (1966) and support the hypothesis (Huxley, 1957, 1980) that muscle tension is produced by simultaneous action of independent force generators, in proportion to the number of myosin bridges overlapped by actin filaments.

Myofilament spacing and force generation in intact frog muscle fibres.

1. The relation between sarcomere length and steady tetanic tension was determined at 10‐12 degrees C for 70‐80 microns long length‐clamped segments of single fibres isolated from the tibialis anterior muscle of the frog, in normal and hypertonic or hypotonic Ringer solutions. 2. The tension depression and potentiation observed in hypertonic and hypotonic Ringers solutions varied with sarcomere length, so that, as opposed to myofilament overlap predictions, the optimum length for tension development was shorter in hypertonic Ringer solution and longer in hypotonic Ringer solution than in normal Ringer solution. As the fibres were stretched from 1.96 to 2.24 microns sarcomere length, both tension depression in hypertonic Ringer solution and tension potentiation in hypotonic Ringer solution increased by 9 and 5%, respectively. 3. Within this range of sarcomere lengths the length‐stiffness relation in hypotonic and in hypertonic Ringer solutions exhibit little or no change relative to that in normal Ringer solution. 4. The results indicate that separation between the thick and the thin myofilaments influences the mechanism of force generation. There is an optimum interfilament distance (10‐12 nm surface to surface between the thick and the thin filaments) for tension production. In isotonic Ringer solution, this corresponds to the interfilament distance at sarcomere lengths around 2.10 microns. The force per attached cross‐bridge, rather than their number, appears to decrease as the interfilament distance is brought above or below the optimum length. Even if this effect is moderate in isotonic Ringer solution, it should be taken into account in models of the force‐generation mechanism.

Force responses to fast ramp stretches in stimulated frog skeletal muscle fibres

Force responses to fast ramp stretches at various velocities were recorded from single muscle fibres isolated from either lumbricalis digiti IV or tibialis anterior muscle of the frog (Rana esculenta) at sarcomere length between 2.15 and 3.25 μm at 15° C. Stretches were applied at rest, at tetanus plateau and during the tetanus rise. Stretches with the same velocity but different accelerations were imposed to the fibre to evaluate the effect of fibre inertia on the force responses. Length changes were measured at sarcomere level with either a laser diffractometer or a striation follower apparatus. The force response to a fast ramp stretch could be divided into two phases. The initial fast one (phase 1) lasts for the acceleration period during which the stretching velocity rises up to the steady state. The second slower phase (phase 2) lasts for the remainder of the stretch and corresponds to the well-known elastic response of the fibre. Most of this paper is concerned with phase 1. The amplitude of the initial fast phase was proportional to the stretching velocity as expected from a viscous response. This viscosity was associated with a very short (about 10 μs) relaxation time. The amplitude of the fast phase increased progressively with tension during the tetanus rise and scaled down with sarcomere length approximately in the same way as tetanic tension and fibre stiffness. These data suggest that activated fibres have a significant internal viscosity which may arise from crossbridge interaction

Crossbridge properties during force enhancement by slow stretching in single intact frog muscle fibres.

The mechanism of force enhancement during lengthening was investigated on single frog muscle fibres by using fast stretches to measure the rupture tension of the crossbridge ensemble. Fast stretches were applied to one end of the activated fibre and force responses were measured at the other. Sarcomere length was measured by a striation follower device. Fast stretching induced a linear increase of tension that reached a peak and fell before the end of the stretch indicating that a sudden increase of fibre compliance occurred due to forced crossbridge detachment induced by the fast loading. The peak tension (critical tension, Pc) and the sarcomere length needed to reach Pc (critical length, Lc) were measured at various tensions during the isometric tetanus rise and during force enhancement by slow lengthening. The data showed that Pc was proportional to the tension generated by the fibre under both isometric and slow lengthening conditions. However, for a given tension increase, Pc was 6.5 times greater during isometric than during lengthening conditions. Isometric critical length was 13.04 ± 0.17 nm per half‐sarcomere (nm hs−1) independently of tension. During slow lengthening critical length fell as the force enhancement increased. For 90% enhancement, Lc reduced to 8.19 ± 0.039 nm hs−1. Assuming that the rupture force of the individual crossbridge is constant, these data indicate that the increase of crossbridge number during lengthening accounts for only 15.4% of the total force enhancement. The remaining 84.6% is accounted for by the increased mean strain of the crossbridges.

Force enhancement after stretch in mammalian muscle fiber: no evidence of cross-bridge involvement

Stretching of activated skeletal muscles induces a force increase above the isometric level persisting after stretch, known as residual force enhancement (RFE). RFE has been extensively studied; nevertheless, its mechanism remains debated. Unlike previous RFE studies, here the excess of force after stretch, termed static tension (ST), was investigated with fast stretches (amplitude: 3-4% sarcomere length; duration: 0.6 ms) applied at low tension during the tetanus rise in fiber bundles from flexor digitorum brevis (FDB) mouse muscle at 30°C. ST was measured at sarcomere length between 2.6 and 4.4 μm in normal and N-benzyl-p-toluene sulphonamide (BTS)-added (10 μM) Tyrode solution. The results showed that ST has the same characteristics and it is equivalent to RFE. ST increased with sarcomere length, reached a peak at 3.5 μm, and decreased to zero at ∼4.5 μm. At 4 μm, where active force was zero, ST was still 50% of maximum. BTS reduced force by ∼75% but had almost no effect on ST. Following stimulation, ST developed earlier than force, with a time course similar to internal Ca(2+) concentration: it was present 1 ms after the stimulus, at zero active force, and peaked at ∼3-ms delay. At 2.7 μm, activation increased the passive sarcomere stiffness by a factor of ∼7 compared with the relaxed state All our data indicate that ST, or RFE, is independent of the cross-bridge presence and it is due to the Ca(2+)-induced stiffening of a sarcomeric structure identifiable with titin.

SARCOMERE TENSION-STIFFNESS RELATION DURING THE TETANUS RISE IN SINGLE FROG MUSCLE FIBRES

The sarcomere stiffness was measured in single muscle fibres during the development of tetanic tension using a method insensitive to fibre intertia and viscosity. The stiffness was calculated by measuring the ratio between tension and sarcomere length during a period of fast sarcomere elongation at constant velocity. Tension changes were corrected for force truncation by the quick recovery mechanism. The results show that the relation between force and stiffness deviates from the direct proportionality less than previously reported. If the deviation is due to the presence of a linear myofilament compliance in series with the cross-bridges, our data suggest that myofilament compliance accounts for about 30% of the sarcomere compliance. This value is significantly smaller than 50–70% determined by X-ray diffraction measurements. These two different findings, however, may be reconciled by assuming that the myofilament compliance is non-linear increasing appropriately at low tension.